There is accumulating evidence indicating that the synthesis and effect of second messengers generated by the blood vessel wall which regulates cholesterol trafficking and proliferative state are altered in atherosclerosis. These second messengers include prostacyclin (PGI/2) and nitric oxide (NO). PGI/2 synthesis is reduced in atherosclerosis, due in part to reduced cyclooxygenase (prostaglandin H synthase, PGHS) expression. Like PGI/2, NO also promotes vasodilation and inhibits smooth muscle cell (SMC) proliferation; its effects are reduced in atherosclerosis. These observations support the premise that PGHS regulation may be linked to NO production. We have preliminary evidence demonstrating that NO promotes eicosanoid generation by two distinct mechanisms: (i) direct activation of PGHS-1, and (ii) upregulation of the expression of mitogen-inducible PGHS-2. The overall goal of this proposal is to determine the relationships between NO and eicosanoid metabolism under normal and lipid-loaded conditions. Experiments proposed in SPECIFIC AIM ONE will determine the mechanism by which NO directly activates PGHS-1. This will be investigated using a variety of biochemical and biophysical techniques, which will be done in part in collaboration with Dr. Silverstein. Next, since PGHS-1 is a membrane- bound enzyme, we hypothesize that PGHS-1 activity is regulated by its lipid environment. Thus, we will evaluate the ability of NO to alter PGHS-1 activity and structure in the presence of polar lipids and cholesterol derived from normal and lipid-enriched SMC. Experiments proposed in SPECIFIC AIM TWO will determine the mechanisms by which NO alters eicosanoid synthesis in intact SMC. PGHS-1 is constitutively expressed, while PGHS-2 is transcriptionally regulated by the binding of NF-kB, AP-1, and SP-1. An important route of PGHS-2 expression occurs through the p21ras signaling cascade, since PDGF which activates cells via p21ras, potently increases PGHS-2 transcription. Since NO also activates NF-kB in a p21ras-dependent manner, NO may induce PGHS-2 expression by this mechanism. Accordingly, experiments are proposed to determine the biochemical mechanisms by which NO alters PGHS-1 activity, as well as PGHS-2 mRNA and protein expression and cellular activity. These studies will be performed in collaboration with Project IV; these collaborations are crucial to successful completion of experiments proposed in Specific Aim 2. The results of these studies should have important implications, since they may suggest a mechanism by which NO may modulate atherosclerotic disease, that is by promoting PGI/2 generation, which in turn may inhibit cholesteryl ester deposition.